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Cross-reactive cellular and humoral immune responses to Salmonella enterica serovars Typhimurium and Enteritidis are associated with protection to heterologous re-challenge
Veterinary Immunology and Immunopathology 114 (2006) 84–93 www.elsevier.com/locate/vetimm
Cross-reactive cellular and humoral immune responses to Salmonella enterica serovars Typhimurium and Enteritidis are associated with protection to heterologous re-challenge R.K. Beal a,*, P. Wigley c, C. Powers a, P.A. Barrow b, A.L. Smith a,* a
b
Division of Immunology, Institute for Animal Health, Compton, Newbury RG20 7NN, UK School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonnington Campus, Loughborough, UK c Department if Veterinary Pathology, University of Liverpool, Leahurst, Neston, UK Received 16 March 2006; received in revised form 7 July 2006; accepted 25 July 2006
Abstract Chickens infected with Salmonella enterica serovars Typhimurium (ST) and Enteritidis (SE) still represent a major source of human food poisoning via consumption of contaminated meat and eggs. Vaccination represents a sustainable approach to control Salmonella in the chicken and the serovar specificity of immunity has the potential to impact on the need for multivalent vaccines. The issue of cross-reactive immune responses and cross-serovar protection was examined in these experiments. Cellular and humoral immune responses were measured by antigen-specific ELISA and splenocyte proliferation assays during primary infections (with ST and SE) and during a second challenge with homologous or heterologous serovars. Primary infection with ST or SE induced strong lymphocyte proliferation and high levels of specific antibody (IgM, IgG and IgA) responses with substantial serovar cross-reactivity. The occurrence of high levels of splenocyte proliferation and strong antibody responses corresponded to the initiation of clearance with both ST and SE. Re-challenge of ST and SE infection-primed chickens with either serovar resulted in significant levels of protection (assessed by bacterial numbers and rate of clearance) with little difference between homologous or heterologous challenge schedules. Relatively low levels of antigen-specific splenocyte proliferation were detected during secondary infection, which may be caused by splenic T cells exiting to the gut. In contrast, the more rapid specific antibody responses (compared with primary infection controls) indicate the development of a secondary antigen-specific adaptive response. The substantial level of cross-protection between serovars and the level of antigenic cross-reactivity indicates the potential for single serovar live vaccines to protect against both group B and D salmonellae. # 2006 Elsevier B.V. All rights reserved. Keywords: Salmonella; Cross-protection; Vaccination; Cross-reactivity; Chicken; Enteric
1. Introduction Food-borne infection by Salmonella enterica through the consumption of contaminated poultry * Corresponding authors at: Enteric Immunology Group, Division of Immunology, Institute for Animal Health, Compton, Newbury RG20 7NN, UK. Tel.: +44 1625 578411; fax: +44 1635 578237. E-mail addresses: [email protected] (R.K. Beal), [email protected] (A.L. Smith). 0165-2427/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2006.07.011
meat and eggs still represents a major public health concern. Human cases of S. enterica comprise approximately 37% of all cases of bacterial food poisoning in England and Wales (Lawson et al., 2003) with 22,941 cases reported in 2003 (although many more cases are believed to go unreported). Of these cases, S. enterica serovars Enteritidis (SE) and Typhimurium (ST) have been most commonly isolated, though a number of other serovars also cause enteritis in man. ST and SE infect chickens via
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the faecal oral route, colonise the alimentary tract of chickens leading to the contamination of meat. In the case of SE, transmission to eggs may also occur and subsequent transmission to humans through consumption of contaminated eggs remains a major public health issue in Europe (Velge et al., 2005). Chickens that become infected when more than a few days old do not generally exhibit clinical symptoms, causing a problem for detection and control in farmed flocks. Changes to legislation in the UK led to a reduction in levels of SE isolated from egg-laying flocks in the late 1990s (Anonymous, 2003) which was attributed to improved hygiene and biosecurity combined with the introduction of vaccination. A concurrent reduction in the number of cases of human salmonellosis was linked to the fall in egg transmission of SE, although, more recently levels seem to have stabilised (Lawson et al., 2003). The need for efficacious control measures, particularly within the broiler industry, remains relevant and drives research focussed on the production of more effective vaccines. Vaccination has been successful in decreasing the incidence of fowl typhoid (caused by S. enterica serovar Gallinarum and S. enterica serovar Pullorum) worldwide and is an attractive method for the control of food-poisoning serovars (Smith, 1956; Silva et al., 1981). Vaccines currently used in the control of SE and ST in Europe are either killed vaccines (such as Salenvac1 and SalenvacT1) or produced from randomly attenuated live strains (such as TAD E1 and TAD T1). The protection offered by the killed vaccines is not as strong as that afforded by live attenuated vaccine strains or wild type S. enterica infections (Barrow et al., 1990; Beal et al., 2004a,b). However, the nature of the attenuation of these vaccines and the immune response they generate are poorly understood. Much of the recent research has focused on the use of rationally attenuated live vaccines, some of which are licensed for use in the USA. Chicks vaccinated with a DNA adenine methylase mutant of ST (Dam UK-1) were partially protected against both ST and SE infection (Dueger et al., 2001). Vaccinations were given orally to 1-day-old chicks, which may have resulted in a competitive exclusion rather than immunological protection (Dueger et al., 2003). Indeed an adaptive response would seem unlikely in chickens vaccinated at this age as they fail to clear wild type enteric ST infection for several weeks and their enteric immune system is still not fully developed at this age (Befus et al., 1980; Gomez Del Moral et al., 1998; Mast
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and Goddeeris, 1999; Bar-Shira et al., 2003; Beal et al., 2004b). Chickens over 3 weeks of age produce a strong humoral and cellular immune response following infection with ST (Beal et al., 2004a,b; Withanage et al., 2005). The antibodies produced in response to infection with either SE or ST have been shown by ELISA to cross-react with heterologous S. enterica serovars (Nicholas and Cullen, 1991; Baay and Huis in ’t Veld, 1993). Whilst it is known that these antibodies have the capacity to cross-react with heterologous serovars, to our knowledge the level of protection to heterologous infection afforded by these cross-reactive immune responses has not been studied. The aim of the work presented here was to examine the serovar specificity of induced immune responses (humoral and cellular) and to determine the level of cross-protection afforded by priming with SE and ST in the chicken. 2. Materials and methods 2.1. Experimental animals Specified pathogen-free (SPF) Rhode Island Red chickens were supplied as 1-day-old chicks by the Poultry Production Unit of the Institute for Animal Health (IAH), Compton Laboratory. Birds were reared in wire cages at 30 8C from 1-day-old, decreasing to 20 8C at 3 weeks of age and given ad libitum access to water and a vegetable-based protein diet (Special Diet Services, Witham, UK). Birds were tagged with wing bands to allow identification of individuals. 2.2. Bacterial strains The infection studies were carried out using spontaneous nalidixic acid resistant (nalr) and spectinomycin resistant (spcr) mutants of ST strain F98 (Phage Type 14) and SE 125589 PT4 that have been used in previous studies (Smith and Tucker, 1975; Barrow et al., 1990, 1998; Barrow and Lovell, 1991). Bacterial cultures were grown from stocks stored in glycerol at 70 8C in Luria Bertani (LB) broth (BD Biosciences, Oxford, UK) at 37 8C in an orbital shaking incubator at 150 rpm. Additionally, spontaneous nalidixic acid resistant mutants of S. gallinarum 9 and S. pullorum 449/87, which have been described previously (Smith, 1956; Wigley et al., 2001), were used to produce soluble antigen preparations.
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Table 1 Summary of the experimental protocol Experimental group 1 2 3 4 5 6
Primary infection (6 weeks) r
S. Typhimurium F98 nal S. Typhimurium F98 nalr S. Enteritidis 125589 PT4 nal r S. Enteritidis 125589 PT4 nal r Uninfected Uninfected
2.3. Experimental protocol Rhode Island Red chickens were given 0.1 ml of an adult gut flora preparation at 1-day-old to minimise variation in the gut flora composition (Barrow et al., 2004). Chickens were randomly assigned into six groups in three rooms; groups 1 and 2 in room 1, groups 3 and 4 in room 2 and groups 5 and 6 in room 3. 2.3.1. Primary infection Chickens were infected at six weeks of age, groups 1 and 2 were infected orally with approx. 4 108 cfu of ST F98 nalr and groups 3 and 4 were infected orally with approx. 4 108 cfu of SE 125589 PT 4 nalr. Groups 5 and 6 remained uninfected as controls. 2.3.2. Secondary infection All groups were (re-)challenged at 14 weeks of age. Groups 1, 3 and 5 were infected orally with approx. 4 108 cfu of ST F98 spcr and groups 2, 4 and 6 were infected orally with approx. 4 108 cfu of SE 125589 PT4 spcr. The infection regimen is summarised in Table 1. 2.4. Enumeration of bacteria from organ samples Organs were removed aseptically in the following order: spleen, liver and caecum at day 13 post-primary infection (dppi) and days 0, 2, 5, and 13 post-second infection (dpsi). Samples were homogenized with phosphate-buffered saline (PBS) and bacterial counts of serial dilutions were made on Brilliant Green (BG) agar (BD Biosciences, Oxford, UK) supplemented with either 1 mg/ml1 novobiocin and 20 mg/ml1 nalidixic acid or 50 mg/ml spectinomycin. Swabs of the cloaca (taken at 6, 13, 20, 27, 35, 41 and 48 dppi) were vortex-mixed in 2 ml of Selenite broth (Oxoid) and plated out onto BG agar (as above), the swabs were incubated overnight at 37 8C and these enriched swabs were again plated onto BG agar. All of the agar plates were incubated overnight at 37 8C before the colonies
Secondary infection (14 weeks) S. S. S. S. S. S.
Typhimurium F98 spcr Enteritidis 125589 PT4 spcr Typhimurium F98 spcr Enteritidis 125589 PT4 spcr Typhimurium F98 spcr Enteritidis 125589 PT4 spcr
were counted (enumeration) or scored as positive or negative pre- and post-enrichment in Selenite broth (swabs). 2.5. Production of soluble Salmonella lysate antigen preparations Preparations of soluble S. enterica lysates were carried out as previously described (Beal et al., 2004a). Briefly, overnight cultures of the serovars described above were used to inoculate 250 ml Erlenmeyer flasks containing 100 ml of LB medium and incubated overnight at 37 8C in an orbital incubator (150 rpm). Bacterial cells were pelleted and washed twice with an equal volume of PBS and re-suspended in 20 ml PBS. The bacterial suspension was subjected to three freeze-thaw cycles in liquid nitrogen before sonication (9 20 s bursts with 1 min cooling between bursts) in 10 ml volumes on ice at an amplitude of 15 mm using a Soniprep 150 (MSE Scientific Instruments, Crawley, UK). The suspension was clarified by centrifugation at 4080 g for 20 min at 4 8C followed by centrifugation at 30,000 g for 20 min at 4 8C to remove insoluble fractions. Protein concentrations of the soluble antigen preparation were measured using the Bradford protein determination kit (Merck, Poole, UK) and aliquots were frozen (20 8C) until used. 2.6. T cell proliferation assays The T cell proliferation assay was performed as described previously (Beal et al., 2004a) from spleen samples taken at 13 dppi and days 2 and 13 dpsi. Briefly, assays were established at 106 splenocytes/ well in U-bottom 96-well microtitre plate and cocultured with RPMI 5% FCS supplemented with either 8.1 mg/ml ST F98 soluble antigen preparation (STAgP), 8.1 mg/ml SE 125589 PT4 (SEAgP) prepared as described previously (Beal et al., 2004a), 200 mg/ml PHA or RPMI-FCS alone in a final volume
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of 200 ml/well. Microtitre plates were incubated at 41 8C in an atmosphere of 5% CO2 in air for 24 h prior to addition of 1 mCi/well of 3H thymidine (Amersham, Little Chalfont, UK) and incubation for a further 18 h. Cells were harvested with a Tomtec Mach IIIM cell harvester (Receptor Technologies, Banbury, UK) and incorporation of 3H thymidine determined with a 1450 Microbeta Trilux scintillation counter (Perkin-Elmer, Beaconsfield, UK). The results were converted to stimulation index (SI) values by the following equation: SI ¼
cpm of stimulated cells : cpm of unstimulated cells
2.7. Enzyme-linked immunosorbent assay (ELISA) Serum was prepared from blood samples of birds taken at 13 dppi and days 0, 3, 5 and 15 dpsi. Levels of serum antibody specific for STAgP or SEAgP were measured by ELISA as described previously (Beal et al., 2004b). Briefly, flat-bottomed 96-well ELISA plates (BD Biosciences, Oxford, UK) were coated with the relevant soluble antigen preparation diluted to 16.2 mg/ml in carbonate/bicarbonate buffer (pH 9.6) overnight at 4 8C then washed with PBS Tween-20 (0.05%) (PBS-T). Following blocking with PBS-T supplemented with 3% skimmed milk powder for 1 h, serum samples were diluted in blocking buffer to 1:400 (for detection of IgM and IgG) and 1:12.5 (IgA), added to the plates and incubated at 37 8C for 1 h. Plates were washed and bound immunoglobulins were detected by incubation at 37 8C for 1 h with horseradish peroxidase conjugated to either goat anti-chicken IgM (1:1000) (Serotec, Oxford, UK), rabbit anti-chicken IgG (1:2000) (Sigma) or goat anti-chicken IgA (1:5000) (Serotec) diluted in blocking buffer. Plates were washed and a solution containing 2,2-azino-di(3-ethylbenzothiazoline-6-sulphonate) (ABTS) and hydrogen peroxide (50 ml/well) was added as the chromagen. The plates were incubated at room temperature in the dark for 30–60 min and the reaction was stopped by addition of 1% SDS. Absorbances were read at 405 nm on a Benchmark microplate reader (Biorad, Hemel Hempstead, UK).
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3. Results 3.1. Primary infection Chickens infected with either SE or ST at 6 weeks of age exhibited a similar pattern of bacterial colonisation and clearance (Fig. 1). Most of the chickens infected with either serovar were Salmonella negative by cloacal swab at 12 dpi (Fig. 1A). However, viable counts from the caecal contents and the spleen demonstrated that all chickens tested were still Salmonella positive at 13 dpi, with similar counts regardless of which serovar was used for the infection (Fig. 1B). Antigen-specific IgG and IgA, and lymphoproliferative responses were detected at 13 dpi in chickens infected with either SE or ST (Figs. 2 and 3). Chickens infected with ST had higher levels of serum IgG specific for both STAgP (P < 0.05) and SEAgP (not statistically significant by t-test) than chickens infected with SE. Both infected groups had significantly higher levels of antigen-reactive serum IgG than the uninfected controls, irrespective of the source of the antigen. In contrast, infection induced the highest serum IgA reactivity against homologous antigen. Nonetheless, birds infected with either serovar demonstrated higher levels of IgA specific for both soluble antigens than uninfected birds.
2.8. Statistics Statistical analysis between groups was carried out using Student’s t-test on Microsoft Excel. Differences between experimental groups were considered significant for P < 0.05.
Fig. 1. (A) Faecal excretion of ST and SE by cloacal swab following oral infection at 6 weeks of age. (B) Viable counts from caecal contents and spleen of chickens 13 dpi with ST or SE. Error bars represent the standard error (n = 5).
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chickens infected with SE was slightly lower than STinfected chickens but still significantly higher than uninfected controls. Splenocytes from chickens infected with either SE or ST exhibited similar proliferation to homologous and heterologous antigen stimulation. 3.2. Secondary infection Chickens were given a homologous or a heterologous re-challenge 55 dppi; in addition primary infections of SE and ST were administered to uninfected age-matched control chickens (primary challenge controls). Chickens that received a primary infection
Fig. 2. STAgP- and SEAgP-specific IgG (A) and IgA (B) from the serum of chickens infected with ST or SE (13 dpi). Error bars represent the standard error (n = 5); groups within columns annotated with different letters differ significantly (P < 0.05).
The components of various S. enterica antigen preparations were separated by SDS-PAGE and the reactivity of serum from ST-infected chickens was examined to assess the nature of the serovar crossreactive response. Antibodies in the serum of STinfected chickens reacted to a large number of polypeptides from soluble antigen preparations of S. Pullorum, S. Gallinarum and SE (data not shown). Splenocytes from chickens infected with ST (13 dppi) proliferated strongly following stimulation by either antigen, exhibiting significantly greater 3H uptake than splenocytes from uninfected chickens (Fig. 3). The level of splenocyte proliferation from
Fig. 3. Proliferation in response to STAgP and SEAgP of splenocytes from chickens infected with ST or SE (13 dpi). Error bars represent the standard error (n = 5); groups within columns annotated with different letters differ significantly (P < 0.05).
Fig. 4. Viable counts of ST and SE (cfu/g) in the caecal contents. Chickens received a priming challenge with SE or ST, or were left unchallenged at 6 weeks of age, and were (re)-challenged with either ST (A) or SE (B) at 14 weeks of age. Error bars represent the standard error (n = 5); groups within columns annotated with different letters differ significantly (P < 0.05), ND = none detected. A summary of the number of birds that were positive for Salmonella at each time point is given in the table.
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Fig. 5. STAgP-specific (A and C) and SEAgP-specific (B and D) serum IgG response. Chickens received a priming challenge with SE or ST, or were left unchallenged at 6 weeks of age, and were (re)-challenged with either ST (A and B) or SE (C and D) at 14 weeks of age. Error bars represent the standard error (n = 5); groups within columns annotated with different letters differ significantly (P < 0.05).
of ST or SE were similarly protected to re-challenge with ST, with reduced viable counts in both the spleen and caecal contents compared with primary challenge controls (Fig. 4). Chickens primed with either SE or ST had reduced numbers of Salmonella in their caecal contents, compared to primary challenge controls after rechallenge with SE. However, the level of immunity was greater in chickens primed with SE than those primed with ST at 5 dpsi (Fig. 4B). All of the chickens given a parallel primary challenge were positive at 5 and 13 dpsi, by comparison many of the birds receiving either a homologous or heterologous re-challenge were culture negative for Salmonella in their caecal contents. The specificity and magnitude of various elements of the immune response were assessed after homologous and heterologous re-challenge. Soon after re-challenge with ST (2 dpsi) higher quantities of ST-specific antibodies (IgG and IgA) were present in chickens primed with ST than those primed with SE or primary challenge controls (Figs. 5A and 6A). By 13 dpsi the STAgP specific IgG and IgA responses were higher in
primary challenge controls than in those primed with ST or SE infection. The IgG response to SEAgP following re-challenge with ST was lower in chickens receiving the homologous re-challenge than in those receiving heterologous re-challenge or primary challenge controls (P < 0.05) (Fig. 5B). The IgA response was low in chickens primed with either serovar in comparison to primary challenge controls at 13 dpsi following ST rechallenge; however, at 2 dpsi chickens primed with SE had a higher IgA response to SEAgP than chickens primed with ST (Fig. 6B). When the second challenge infection was SE, levels of SEAgP-specific IgG and IgA were increased in chickens irrespective of their previous infection history at 2 dpsi; although the highest levels of IgA were observed with serum from primary challenge control chickens at 13 dpsi (P < 0.05) (Figs. 5D and 6D). Levels of serum IgG specific for STAgP were low following re-challenge with SE regardless of the priming infection; however, levels at 2 dpsi were significantly higher than in primary challenge controls (P < 0.05) (Fig. 5C). By 13 dpsi the response to STAgP
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Fig. 6. STAgP-specific (A and C) and SEAgP-specific (B and D) serum IgA response. Chickens received a priming challenge with SE or ST, or were left unchallenged at 6 weeks of age, and were (re)-challenged with either ST (A and B) or SE (C and D) at 14 weeks of age. Error bars represent the standard error (n = 5); groups within columns annotated with different letters differ significantly (P < 0.05).
was low in those receiving homologous re-challenge; but by comparison, was significantly higher in those receiving heterologous re-challenge or in primary challenge controls. The IgA response to STAgP was greater in chickens after heterologous than homologous SE re-challenge, confirming the results seen during the primary infection that the response to STAgP was higher in chickens that had received a ST infection (Fig. 6C). Significant differences in antigen-specific splenocyte proliferation were only evident at 2 dpsi (Fig. 7). Following ST re-challenge, splenocyte proliferation in response to both SEAgP and STAgP were significantly higher in those receiving a homologous or heterologous re-challenge compared to age matched primary challenge controls (Fig. 7A and B). Whilst there appeared to be a slight increase in the proliferation of splenocytes from chickens receiving a homologous SE re-challenge at 2 dpsi, this proliferation was not significantly different to those from primary challenge
controls (Fig. 7C and D). At 13 dpsi all of the responses between the various groups were comparable. 4. Discussion The use of a vaccine to protect chickens from a broad range of S. enterica serovars would be a valuable tool for producers of chicken meat and eggs. Challenge in the face of an already existing primary infection (vaccine) presents a problem for data interpretation, where reduced infection may be a result of immune protection or competitive exclusion by the ‘‘vaccinating’’ strain. The immune system of the chickens is still not fully developed and so attenuated strains can persist for long periods in chicks vaccinated at 1-day-old (Barbezange et al., 2000). Current research has focused on potential live attenuated strains such as the Dam and aroA mutants (Cooper et al., 1994; Dueger et al., 2001), which conferred varying levels of protection to challenge with homologous and heterologous virulent
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Fig. 7. Proliferation of splenocytes in response to STAgP and SEAgP. Chickens received a priming challenge with SE or ST, or were left unchallenged at 6 weeks of age, and were (re)-challenged with either ST (A and B) or SE (C and D) at 14 weeks of age. Error bars represent the standard error (n = 5); groups within columns annotated with different letters differ significantly (P < 0.05).
serovars. Vaccination in these studies was undertaken shortly after hatch and therefore reduced infection may result from competitive exclusion rather than immune protection. Indeed studies by Methner et al. (2001) looked specifically at using a competitive exclusion culture alongside a live ST vaccine in 1-day-old chicks; they observed no increase in protection against a virulent strain in those receiving both treatments to those treated with the competitive exclusion culture alone. In the present study we demonstrated the ability of virulent S. enterica serovars to stimulate broadly reactive cellular and humoral immune responses to antigen preparations derived from different S. enterica serovars in immunologically mature chickens. We also observed protection to re-challenge with heterologous or homologous serovars, which corresponded with a high level of cross-reactivity of the induced immune responses to both serovars of S. enterica. It is possible that an ‘‘adjuvant effect’’ (i.e. non-specific stimulation of the immune system by molecules such as Toll-like
receptor agonists) conferred by the priming challenge could contribute to protection against secondary challenge. However, the protection observed in the present study occurred following a challenge 8 weeks after the initial infection and as such is unlikely to be a result of an ‘‘adjuvant effect’’ which would be expected to be much more transient. The high level of cross-reactivity of antibodies to antigen produced from different serovars for both IgA and IgG was not unexpected and is consistent with previous reports (Hassan et al., 1990; Nicholas and Cullen, 1991; Barrow et al., 1992). Birds infected with ST also had higher levels of serum IgG specific for SE antigens than those birds infected with SE. Unlike IgA, IgG is only present in systemic sites; therefore, greater induction of Salmonella-specific IgG by ST could be a response to a higher level of systemic infection. However, levels of Salmonella in the spleen were similar regardless of infecting serovar at 14 dpi, although higher numbers may have been present at
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earlier time points following infection. Cross-reactivity of serum IgA to heterologous serovars was also high, although the IgA response to homologous antigen was consistently highest. Due to the largely enteric nature of the pathogen, IgA is more likely to be involved in any protective immune response after transport into the gut lumen (Lebacq-Verheyden et al., 1972; Leslie and Martin, 1973; Berthelot-Herault et al., 2003). However, whilst the IgA response appears to be more serovar specific than the production of IgG, protection to rechallenge with ST was similar regardless of the priming serovar. The results from the present study indicate a large degree of serovar cross-reactivity in the adaptive immune responses of the chicken to primary infection with either serovar of S. enterica. The data from this study is consistent with previous findings in our laboratory that splenocytes from chickens infected with ST are capable of proliferating in response to SE, SG and SP in addition to ST (unpublished data). Very few studies have examined the capacity of lymphocytes from S. enterica infected chickens to respond to S. enterica antigens ex vivo. Work from our laboratory demonstrated the capacity of lymphocytes to respond to ex vivo antigen stimulation following primary but not secondary infection (Beal et al., 2004a). The data presented here also shows a lack of splenic T cell proliferation following re-infection with either homologous or heterologous serovars, which may be interpreted as the exit of responding T cells from the spleen to sites of infection. The degree of cross-protection acheived by ST to subsequent SE infection was close to that afforded by homologous re-challenge in terms of both systemic and enteric infection. In contrast, SE was less effective in protecting against subsequent ST re-challenge with slightly higher enteric counts than chickens receiving a homologous ST re-challenge. However, the very low numbers of Salmonella in the spleens of both groups indicate that both serovars cross-protect equally well at systemic sites. The capacity of ST and SE to protect against heterologous re-challenge was reflected in the antibody response following secondary infection. High cross-reactivity in serum IgA and IgG against SEAgP and STAgP was evident for all of the infection protocols employed. Whilst high levels of circulating IgA and IgG were observed following challenge and re-challenge, our recent studies suggest that neither B cells or antibody are essential for clearance of a primary or secondary infection with ST (Beal et al., 2006), although no such data exists for primary, heterologous or homologous SE infections.
The data presented here demonstrates that S. enterica from serovar group B can protect against re-challenge with group D and vice versa in immunologically mature chickens. Whilst these data are encouraging for the use of single serovar vaccines to protect mature egg layers against a range of infective serovars, broiler chickens may not develop sufficient immunity in their short production lives for any significant cross-protection to result. Previous work (Beal et al., 2004b) suggests that humoral and cellular immune responses to Salmonella Typhimurium are very low and increase with age (up to 6 weeks). This corresponds with maturation of lymphoid aggregates and immune cell subsets (such as T cells, B cells and heterophils) over the same time period (Befus et al., 1980; Vervelde and Jeurissen, 1993; Gomez Del Moral et al., 1998; Wells et al., 1998; BarShira et al., 2003). Whilst live vaccine strains have shown high levels of protection against infection with heterologous serovars, it has not been shown if this is mediated by an immune response or by competitive exclusion (Dueger et al., 2001, 2003; Methner et al., 2001). The data presented here indicate that there is sufficient immunological cross-protection to merit further investigation of the possibility of developing single serovar live vaccines to protect against both group B and D Salmonella serovars. Acknowledgements We thank the staff of the production and experimental units of the IAH and the Biotechnology and Biological Sciences Research Council and DEFRAHEFCE for funding this research (grant nos. 8/ BFP11365 and VT-0104). References Anonymous, 2003. Postnote 193: Food poisoning. Parliamentary Office of Science and Technology. Baay, M.F., Huis in ’t Veld, J.H., 1993. Alternative antigens reduce cross-reactions in an ELISA for the detection of Salmonella enteritidis in poultry. J. Appl. Bacteriol. 74, 243–247. Bar-Shira, E., Sklan, D., Friedman, A., 2003. Establishment of immune competence in the avian GALT during the immediate post-hatch period. Dev. Comp. Immunol. 27, 147–157. Barbezange, C., Humbert, F., Rose, V., Lalande, F., Salvat, G., 2000. Some safety aspects of salmonella vaccines for poultry: distribution and persistence of three Salmonella typhimurium live vaccines. Avian Dis. 44, 968–976. Barrow, P.A., Berchieri Jr., A., al-Haddad, O., 1992. Serological response of chickens to infection with Salmonella gallinarum– S. pullorum detected by enzyme-linked immunosorbent assay. Avian Dis. 36, 227–236. Barrow, P.A., Bumstead, N., Marston, K., Lovell, M.A., Wigley, P., 2004. Faecal shedding and intestinal colonization of Salmonella
R.K. Beal et al. / Veterinary Immunology and Immunopathology 114 (2006) 84–93 enterica in in-bred chickens: the effect of host-genetic background. Epidemiol. Infect. 132, 117–126. Barrow, P.A., Hassan, J.O., Berchieri Jr., A., 1990. Reduction in faecal excretion of Salmonella typhimurium strain F98 in chickens vaccinated with live and killed S. typhimurium organisms. Epidemiol. Infect. 104, 413–426. Barrow, P.A., Lovell, M.A., 1991. Experimental infection of egglaying hens with Salmonella enteritidis. Avian Pathol. 20, 339– 352. Barrow, P.A., Lovell, M.A., Szmolleny, G., Murphy, C.K., 1998. Effect of enrofloxacin administration on excretion of Salmonella enteritidis by experimentally infected chickens and on quinolone resistance of their Esherichia coli flora. Avian Pathol. 27, 586– 590. Beal, R.K., Powers, C., Davison, T.F., Barrow, P.A., Smith, A.L., 2006. Clearance of enteric Salmonella enterica serovar typhimurium in chickens is independent of B-cell function. Infect. Immun. 74, 1442–1444. Beal, R.K., Powers, C., Wigley, P., Barrow, P.A., Smith, A.L., 2004a. Temporal dynamics of the cellular, humoral and cytokine responses in chickens during primary and secondary infection with Salmonella enterica serovar typhimurium. Avian Pathol. 33, 25–33. Beal, R.K., Wigley, P., Powers, C., Hulme, S.D., Barrow, P.A., Smith, A.L., 2004b. Age at primary infection with Salmonella enterica serovar typhimurium in the chicken influences persistence of infection and subsequent immunity to re-challenge. Vet. Immunol. Immunopathol. 100, 151–164. Befus, A.D., Johnston, N., Leslie, G.A., Bienenstock, J., 1980. Gutassociated lymphoid tissue in the chicken. Part I. Morphology, ontogeny, and some functional characteristics of Peyer’s patches. J. Immunol. 125, 2626–2632. Berthelot-Herault, F., Mompart, F., Zygmunt, M.S., Dubray, G., Duchet-Suchaux, M., 2003. Antibody responses in the serum and gut of chicken lines differing in cecal carriage of Salmonella enteritidis. Vet. Immunol. Immunopathol. 96, 43–52. Cooper, G.L., Venables, L.M., Woodward, M.J., Hormaeche, C.E., 1994. Vaccination of chickens with strain CVL30, a genetically defined Salmonella enteritidis aroA live oral vaccine candidate. Infect. Immun. 62, 4747–4754. Dueger, E.L., House, J.K., Heithoff, D.M., Mahan, M.J., 2001. Salmonella DNA adenine methylase mutants elicit protective immune responses to homologous and heterologous serovars in chickens. Infect. Immun. 69, 7950–7954. Dueger, E.L., House, J.K., Heithoff, D.M., Mahan, M.J., 2003. Salmonella DNA adenine methylase mutants prevent colonization of newly hatched chickens by homologous and heterologous serovars. Int. J. Food Microbiol. 80, 153–159. Gomez Del Moral, M., Fonfria, J., Varas, A., Jimenez, E., Moreno, J., Zapata, A.G., 1998. Appearance and development of lymphoid cells in the chicken (Gallus gallus) caecal tonsil. Anat. Rec. 250, 182–189.
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Hassan, J.O., Barrow, P.A., Mockett, A.P., McLeod, S., 1990. Antibody response to experimental Salmonella typhimurium infection in chickens measured by ELISA. Vet. Rec. 126, 519–522. Lawson, A., McLauchin, J., Smith, H., Threllfall, J., 2003. Department of Gastrointestinal Infections Annual Report. Health Protection Agency. Lebacq-Verheyden, A.M., Vaerman, J.P., Heremans, J.F., 1972. A possible homologue of mammalian IgA in chicken serum and secretions. Immunology 22, 165–175. Leslie, G.A., Martin, L.N., 1973. Studies on the secretory immunologic system of fowl. Part 3. Serum and secretory IgA of the chicken. J. Immunol. 110, 1–9. Mast, J., Goddeeris, B.M., 1999. Development of immunocompetence of broiler chickens. Vet. Immunol. Immunopathol. 70, 245–256. Methner, U., Berndt, A., Steinbach, G., 2001. Combination of competitive exclusion and immunization with an attenuated live Salmonella vaccine strain in chickens. Avian Dis. 45, 631– 638. Nicholas, R.A., Cullen, G.A., 1991. Development and application of an ELISA for detecting antibodies to Salmonella enteritidis in chicken flocks. Vet. Rec. 128, 74–76. Silva, E.N., Snoeyenbos, G.H., Weinack, O.M., Smyser, C.F., 1981. The influence of native gut microflora on the colonization and infection of Salmonella gallinarum in chickens. Avian Dis. 25, 68–73. Smith, H.W., 1956. The use of live vaccines in experimental Salmonella gallinarum infection in chickens with observations on their interference effect. J. Hyg. (Lond.) 54, 419–432. Smith, H.W., Tucker, J.F., 1975. The effect of antibiotic therapy on the faecal excretion of Salmonella typhimurium by experimentally infected chickens. J. Hyg. (Lond.) 75, 275–292. Velge, P., Cloeckaert, A., Barrow, P., 2005. Emergence of Salmonella epidemics: the problems related to Salmonella enterica serotype enteritidis and multiple antibiotic resistance in other major serotypes. Vet. Res. 36, 267–288. Vervelde, L., Jeurissen, S.H., 1993. Postnatal development of intraepithelial leukocytes in the chicken digestive tract: phenotypical characterization in situ. Cell Tissue Res. 274, 295–301. Wells, L.L., Lowry, V.K., DeLoach, J.R., Kogut, M.H., 1998. Agedependent phagocytosis and bactericidal activities of the chicken heterophil. Dev. Comp. Immunol. 22, 103–109. Wigley, P., Berchieri Jr., A., Page, K.L., Smith, A.L., Barrow, P.A., 2001. Salmonella enterica serovar pullorum persists in splenic macrophages and in the reproductive tract during persistent, disease-free carriage in chickens. Infect. Immun. 69, 7873– 7879. Withanage, G.S., Wigley, P., Kaiser, P., Mastroeni, P., Brooks, H., Powers, C., Beal, R., Barrow, P., Maskell, D., McConnell, I., 2005. Cytokine and chemokine responses associated with clearance of a primary Salmonella enterica serovar typhimurium infection in the chicken and in protective immunity to rechallenge. Infect. Immun. 73, 5173–5182.
Cross-reactive cellular and humoral immune responses to Salmonella enterica serovars Typhimurium and Enteritidis are associated with protection to heterologous re-challenge R.K. Beal a,*, P. Wigley c, C. Powers a, P.A. Barrow b, A.L. Smith a,* a
b
Division of Immunology, Institute for Animal Health, Compton, Newbury RG20 7NN, UK School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonnington Campus, Loughborough, UK c Department if Veterinary Pathology, University of Liverpool, Leahurst, Neston, UK Received 16 March 2006; received in revised form 7 July 2006; accepted 25 July 2006
Abstract Chickens infected with Salmonella enterica serovars Typhimurium (ST) and Enteritidis (SE) still represent a major source of human food poisoning via consumption of contaminated meat and eggs. Vaccination represents a sustainable approach to control Salmonella in the chicken and the serovar specificity of immunity has the potential to impact on the need for multivalent vaccines. The issue of cross-reactive immune responses and cross-serovar protection was examined in these experiments. Cellular and humoral immune responses were measured by antigen-specific ELISA and splenocyte proliferation assays during primary infections (with ST and SE) and during a second challenge with homologous or heterologous serovars. Primary infection with ST or SE induced strong lymphocyte proliferation and high levels of specific antibody (IgM, IgG and IgA) responses with substantial serovar cross-reactivity. The occurrence of high levels of splenocyte proliferation and strong antibody responses corresponded to the initiation of clearance with both ST and SE. Re-challenge of ST and SE infection-primed chickens with either serovar resulted in significant levels of protection (assessed by bacterial numbers and rate of clearance) with little difference between homologous or heterologous challenge schedules. Relatively low levels of antigen-specific splenocyte proliferation were detected during secondary infection, which may be caused by splenic T cells exiting to the gut. In contrast, the more rapid specific antibody responses (compared with primary infection controls) indicate the development of a secondary antigen-specific adaptive response. The substantial level of cross-protection between serovars and the level of antigenic cross-reactivity indicates the potential for single serovar live vaccines to protect against both group B and D salmonellae. # 2006 Elsevier B.V. All rights reserved. Keywords: Salmonella; Cross-protection; Vaccination; Cross-reactivity; Chicken; Enteric
1. Introduction Food-borne infection by Salmonella enterica through the consumption of contaminated poultry * Corresponding authors at: Enteric Immunology Group, Division of Immunology, Institute for Animal Health, Compton, Newbury RG20 7NN, UK. Tel.: +44 1625 578411; fax: +44 1635 578237. E-mail addresses: [email protected] (R.K. Beal), [email protected] (A.L. Smith). 0165-2427/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2006.07.011
meat and eggs still represents a major public health concern. Human cases of S. enterica comprise approximately 37% of all cases of bacterial food poisoning in England and Wales (Lawson et al., 2003) with 22,941 cases reported in 2003 (although many more cases are believed to go unreported). Of these cases, S. enterica serovars Enteritidis (SE) and Typhimurium (ST) have been most commonly isolated, though a number of other serovars also cause enteritis in man. ST and SE infect chickens via
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the faecal oral route, colonise the alimentary tract of chickens leading to the contamination of meat. In the case of SE, transmission to eggs may also occur and subsequent transmission to humans through consumption of contaminated eggs remains a major public health issue in Europe (Velge et al., 2005). Chickens that become infected when more than a few days old do not generally exhibit clinical symptoms, causing a problem for detection and control in farmed flocks. Changes to legislation in the UK led to a reduction in levels of SE isolated from egg-laying flocks in the late 1990s (Anonymous, 2003) which was attributed to improved hygiene and biosecurity combined with the introduction of vaccination. A concurrent reduction in the number of cases of human salmonellosis was linked to the fall in egg transmission of SE, although, more recently levels seem to have stabilised (Lawson et al., 2003). The need for efficacious control measures, particularly within the broiler industry, remains relevant and drives research focussed on the production of more effective vaccines. Vaccination has been successful in decreasing the incidence of fowl typhoid (caused by S. enterica serovar Gallinarum and S. enterica serovar Pullorum) worldwide and is an attractive method for the control of food-poisoning serovars (Smith, 1956; Silva et al., 1981). Vaccines currently used in the control of SE and ST in Europe are either killed vaccines (such as Salenvac1 and SalenvacT1) or produced from randomly attenuated live strains (such as TAD E1 and TAD T1). The protection offered by the killed vaccines is not as strong as that afforded by live attenuated vaccine strains or wild type S. enterica infections (Barrow et al., 1990; Beal et al., 2004a,b). However, the nature of the attenuation of these vaccines and the immune response they generate are poorly understood. Much of the recent research has focused on the use of rationally attenuated live vaccines, some of which are licensed for use in the USA. Chicks vaccinated with a DNA adenine methylase mutant of ST (Dam UK-1) were partially protected against both ST and SE infection (Dueger et al., 2001). Vaccinations were given orally to 1-day-old chicks, which may have resulted in a competitive exclusion rather than immunological protection (Dueger et al., 2003). Indeed an adaptive response would seem unlikely in chickens vaccinated at this age as they fail to clear wild type enteric ST infection for several weeks and their enteric immune system is still not fully developed at this age (Befus et al., 1980; Gomez Del Moral et al., 1998; Mast
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and Goddeeris, 1999; Bar-Shira et al., 2003; Beal et al., 2004b). Chickens over 3 weeks of age produce a strong humoral and cellular immune response following infection with ST (Beal et al., 2004a,b; Withanage et al., 2005). The antibodies produced in response to infection with either SE or ST have been shown by ELISA to cross-react with heterologous S. enterica serovars (Nicholas and Cullen, 1991; Baay and Huis in ’t Veld, 1993). Whilst it is known that these antibodies have the capacity to cross-react with heterologous serovars, to our knowledge the level of protection to heterologous infection afforded by these cross-reactive immune responses has not been studied. The aim of the work presented here was to examine the serovar specificity of induced immune responses (humoral and cellular) and to determine the level of cross-protection afforded by priming with SE and ST in the chicken. 2. Materials and methods 2.1. Experimental animals Specified pathogen-free (SPF) Rhode Island Red chickens were supplied as 1-day-old chicks by the Poultry Production Unit of the Institute for Animal Health (IAH), Compton Laboratory. Birds were reared in wire cages at 30 8C from 1-day-old, decreasing to 20 8C at 3 weeks of age and given ad libitum access to water and a vegetable-based protein diet (Special Diet Services, Witham, UK). Birds were tagged with wing bands to allow identification of individuals. 2.2. Bacterial strains The infection studies were carried out using spontaneous nalidixic acid resistant (nalr) and spectinomycin resistant (spcr) mutants of ST strain F98 (Phage Type 14) and SE 125589 PT4 that have been used in previous studies (Smith and Tucker, 1975; Barrow et al., 1990, 1998; Barrow and Lovell, 1991). Bacterial cultures were grown from stocks stored in glycerol at 70 8C in Luria Bertani (LB) broth (BD Biosciences, Oxford, UK) at 37 8C in an orbital shaking incubator at 150 rpm. Additionally, spontaneous nalidixic acid resistant mutants of S. gallinarum 9 and S. pullorum 449/87, which have been described previously (Smith, 1956; Wigley et al., 2001), were used to produce soluble antigen preparations.
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Table 1 Summary of the experimental protocol Experimental group 1 2 3 4 5 6
Primary infection (6 weeks) r
S. Typhimurium F98 nal S. Typhimurium F98 nalr S. Enteritidis 125589 PT4 nal r S. Enteritidis 125589 PT4 nal r Uninfected Uninfected
2.3. Experimental protocol Rhode Island Red chickens were given 0.1 ml of an adult gut flora preparation at 1-day-old to minimise variation in the gut flora composition (Barrow et al., 2004). Chickens were randomly assigned into six groups in three rooms; groups 1 and 2 in room 1, groups 3 and 4 in room 2 and groups 5 and 6 in room 3. 2.3.1. Primary infection Chickens were infected at six weeks of age, groups 1 and 2 were infected orally with approx. 4 108 cfu of ST F98 nalr and groups 3 and 4 were infected orally with approx. 4 108 cfu of SE 125589 PT 4 nalr. Groups 5 and 6 remained uninfected as controls. 2.3.2. Secondary infection All groups were (re-)challenged at 14 weeks of age. Groups 1, 3 and 5 were infected orally with approx. 4 108 cfu of ST F98 spcr and groups 2, 4 and 6 were infected orally with approx. 4 108 cfu of SE 125589 PT4 spcr. The infection regimen is summarised in Table 1. 2.4. Enumeration of bacteria from organ samples Organs were removed aseptically in the following order: spleen, liver and caecum at day 13 post-primary infection (dppi) and days 0, 2, 5, and 13 post-second infection (dpsi). Samples were homogenized with phosphate-buffered saline (PBS) and bacterial counts of serial dilutions were made on Brilliant Green (BG) agar (BD Biosciences, Oxford, UK) supplemented with either 1 mg/ml1 novobiocin and 20 mg/ml1 nalidixic acid or 50 mg/ml spectinomycin. Swabs of the cloaca (taken at 6, 13, 20, 27, 35, 41 and 48 dppi) were vortex-mixed in 2 ml of Selenite broth (Oxoid) and plated out onto BG agar (as above), the swabs were incubated overnight at 37 8C and these enriched swabs were again plated onto BG agar. All of the agar plates were incubated overnight at 37 8C before the colonies
Secondary infection (14 weeks) S. S. S. S. S. S.
Typhimurium F98 spcr Enteritidis 125589 PT4 spcr Typhimurium F98 spcr Enteritidis 125589 PT4 spcr Typhimurium F98 spcr Enteritidis 125589 PT4 spcr
were counted (enumeration) or scored as positive or negative pre- and post-enrichment in Selenite broth (swabs). 2.5. Production of soluble Salmonella lysate antigen preparations Preparations of soluble S. enterica lysates were carried out as previously described (Beal et al., 2004a). Briefly, overnight cultures of the serovars described above were used to inoculate 250 ml Erlenmeyer flasks containing 100 ml of LB medium and incubated overnight at 37 8C in an orbital incubator (150 rpm). Bacterial cells were pelleted and washed twice with an equal volume of PBS and re-suspended in 20 ml PBS. The bacterial suspension was subjected to three freeze-thaw cycles in liquid nitrogen before sonication (9 20 s bursts with 1 min cooling between bursts) in 10 ml volumes on ice at an amplitude of 15 mm using a Soniprep 150 (MSE Scientific Instruments, Crawley, UK). The suspension was clarified by centrifugation at 4080 g for 20 min at 4 8C followed by centrifugation at 30,000 g for 20 min at 4 8C to remove insoluble fractions. Protein concentrations of the soluble antigen preparation were measured using the Bradford protein determination kit (Merck, Poole, UK) and aliquots were frozen (20 8C) until used. 2.6. T cell proliferation assays The T cell proliferation assay was performed as described previously (Beal et al., 2004a) from spleen samples taken at 13 dppi and days 2 and 13 dpsi. Briefly, assays were established at 106 splenocytes/ well in U-bottom 96-well microtitre plate and cocultured with RPMI 5% FCS supplemented with either 8.1 mg/ml ST F98 soluble antigen preparation (STAgP), 8.1 mg/ml SE 125589 PT4 (SEAgP) prepared as described previously (Beal et al., 2004a), 200 mg/ml PHA or RPMI-FCS alone in a final volume
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of 200 ml/well. Microtitre plates were incubated at 41 8C in an atmosphere of 5% CO2 in air for 24 h prior to addition of 1 mCi/well of 3H thymidine (Amersham, Little Chalfont, UK) and incubation for a further 18 h. Cells were harvested with a Tomtec Mach IIIM cell harvester (Receptor Technologies, Banbury, UK) and incorporation of 3H thymidine determined with a 1450 Microbeta Trilux scintillation counter (Perkin-Elmer, Beaconsfield, UK). The results were converted to stimulation index (SI) values by the following equation: SI ¼
cpm of stimulated cells : cpm of unstimulated cells
2.7. Enzyme-linked immunosorbent assay (ELISA) Serum was prepared from blood samples of birds taken at 13 dppi and days 0, 3, 5 and 15 dpsi. Levels of serum antibody specific for STAgP or SEAgP were measured by ELISA as described previously (Beal et al., 2004b). Briefly, flat-bottomed 96-well ELISA plates (BD Biosciences, Oxford, UK) were coated with the relevant soluble antigen preparation diluted to 16.2 mg/ml in carbonate/bicarbonate buffer (pH 9.6) overnight at 4 8C then washed with PBS Tween-20 (0.05%) (PBS-T). Following blocking with PBS-T supplemented with 3% skimmed milk powder for 1 h, serum samples were diluted in blocking buffer to 1:400 (for detection of IgM and IgG) and 1:12.5 (IgA), added to the plates and incubated at 37 8C for 1 h. Plates were washed and bound immunoglobulins were detected by incubation at 37 8C for 1 h with horseradish peroxidase conjugated to either goat anti-chicken IgM (1:1000) (Serotec, Oxford, UK), rabbit anti-chicken IgG (1:2000) (Sigma) or goat anti-chicken IgA (1:5000) (Serotec) diluted in blocking buffer. Plates were washed and a solution containing 2,2-azino-di(3-ethylbenzothiazoline-6-sulphonate) (ABTS) and hydrogen peroxide (50 ml/well) was added as the chromagen. The plates were incubated at room temperature in the dark for 30–60 min and the reaction was stopped by addition of 1% SDS. Absorbances were read at 405 nm on a Benchmark microplate reader (Biorad, Hemel Hempstead, UK).
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3. Results 3.1. Primary infection Chickens infected with either SE or ST at 6 weeks of age exhibited a similar pattern of bacterial colonisation and clearance (Fig. 1). Most of the chickens infected with either serovar were Salmonella negative by cloacal swab at 12 dpi (Fig. 1A). However, viable counts from the caecal contents and the spleen demonstrated that all chickens tested were still Salmonella positive at 13 dpi, with similar counts regardless of which serovar was used for the infection (Fig. 1B). Antigen-specific IgG and IgA, and lymphoproliferative responses were detected at 13 dpi in chickens infected with either SE or ST (Figs. 2 and 3). Chickens infected with ST had higher levels of serum IgG specific for both STAgP (P < 0.05) and SEAgP (not statistically significant by t-test) than chickens infected with SE. Both infected groups had significantly higher levels of antigen-reactive serum IgG than the uninfected controls, irrespective of the source of the antigen. In contrast, infection induced the highest serum IgA reactivity against homologous antigen. Nonetheless, birds infected with either serovar demonstrated higher levels of IgA specific for both soluble antigens than uninfected birds.
2.8. Statistics Statistical analysis between groups was carried out using Student’s t-test on Microsoft Excel. Differences between experimental groups were considered significant for P < 0.05.
Fig. 1. (A) Faecal excretion of ST and SE by cloacal swab following oral infection at 6 weeks of age. (B) Viable counts from caecal contents and spleen of chickens 13 dpi with ST or SE. Error bars represent the standard error (n = 5).
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chickens infected with SE was slightly lower than STinfected chickens but still significantly higher than uninfected controls. Splenocytes from chickens infected with either SE or ST exhibited similar proliferation to homologous and heterologous antigen stimulation. 3.2. Secondary infection Chickens were given a homologous or a heterologous re-challenge 55 dppi; in addition primary infections of SE and ST were administered to uninfected age-matched control chickens (primary challenge controls). Chickens that received a primary infection
Fig. 2. STAgP- and SEAgP-specific IgG (A) and IgA (B) from the serum of chickens infected with ST or SE (13 dpi). Error bars represent the standard error (n = 5); groups within columns annotated with different letters differ significantly (P < 0.05).
The components of various S. enterica antigen preparations were separated by SDS-PAGE and the reactivity of serum from ST-infected chickens was examined to assess the nature of the serovar crossreactive response. Antibodies in the serum of STinfected chickens reacted to a large number of polypeptides from soluble antigen preparations of S. Pullorum, S. Gallinarum and SE (data not shown). Splenocytes from chickens infected with ST (13 dppi) proliferated strongly following stimulation by either antigen, exhibiting significantly greater 3H uptake than splenocytes from uninfected chickens (Fig. 3). The level of splenocyte proliferation from
Fig. 3. Proliferation in response to STAgP and SEAgP of splenocytes from chickens infected with ST or SE (13 dpi). Error bars represent the standard error (n = 5); groups within columns annotated with different letters differ significantly (P < 0.05).
Fig. 4. Viable counts of ST and SE (cfu/g) in the caecal contents. Chickens received a priming challenge with SE or ST, or were left unchallenged at 6 weeks of age, and were (re)-challenged with either ST (A) or SE (B) at 14 weeks of age. Error bars represent the standard error (n = 5); groups within columns annotated with different letters differ significantly (P < 0.05), ND = none detected. A summary of the number of birds that were positive for Salmonella at each time point is given in the table.
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Fig. 5. STAgP-specific (A and C) and SEAgP-specific (B and D) serum IgG response. Chickens received a priming challenge with SE or ST, or were left unchallenged at 6 weeks of age, and were (re)-challenged with either ST (A and B) or SE (C and D) at 14 weeks of age. Error bars represent the standard error (n = 5); groups within columns annotated with different letters differ significantly (P < 0.05).
of ST or SE were similarly protected to re-challenge with ST, with reduced viable counts in both the spleen and caecal contents compared with primary challenge controls (Fig. 4). Chickens primed with either SE or ST had reduced numbers of Salmonella in their caecal contents, compared to primary challenge controls after rechallenge with SE. However, the level of immunity was greater in chickens primed with SE than those primed with ST at 5 dpsi (Fig. 4B). All of the chickens given a parallel primary challenge were positive at 5 and 13 dpsi, by comparison many of the birds receiving either a homologous or heterologous re-challenge were culture negative for Salmonella in their caecal contents. The specificity and magnitude of various elements of the immune response were assessed after homologous and heterologous re-challenge. Soon after re-challenge with ST (2 dpsi) higher quantities of ST-specific antibodies (IgG and IgA) were present in chickens primed with ST than those primed with SE or primary challenge controls (Figs. 5A and 6A). By 13 dpsi the STAgP specific IgG and IgA responses were higher in
primary challenge controls than in those primed with ST or SE infection. The IgG response to SEAgP following re-challenge with ST was lower in chickens receiving the homologous re-challenge than in those receiving heterologous re-challenge or primary challenge controls (P < 0.05) (Fig. 5B). The IgA response was low in chickens primed with either serovar in comparison to primary challenge controls at 13 dpsi following ST rechallenge; however, at 2 dpsi chickens primed with SE had a higher IgA response to SEAgP than chickens primed with ST (Fig. 6B). When the second challenge infection was SE, levels of SEAgP-specific IgG and IgA were increased in chickens irrespective of their previous infection history at 2 dpsi; although the highest levels of IgA were observed with serum from primary challenge control chickens at 13 dpsi (P < 0.05) (Figs. 5D and 6D). Levels of serum IgG specific for STAgP were low following re-challenge with SE regardless of the priming infection; however, levels at 2 dpsi were significantly higher than in primary challenge controls (P < 0.05) (Fig. 5C). By 13 dpsi the response to STAgP
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Fig. 6. STAgP-specific (A and C) and SEAgP-specific (B and D) serum IgA response. Chickens received a priming challenge with SE or ST, or were left unchallenged at 6 weeks of age, and were (re)-challenged with either ST (A and B) or SE (C and D) at 14 weeks of age. Error bars represent the standard error (n = 5); groups within columns annotated with different letters differ significantly (P < 0.05).
was low in those receiving homologous re-challenge; but by comparison, was significantly higher in those receiving heterologous re-challenge or in primary challenge controls. The IgA response to STAgP was greater in chickens after heterologous than homologous SE re-challenge, confirming the results seen during the primary infection that the response to STAgP was higher in chickens that had received a ST infection (Fig. 6C). Significant differences in antigen-specific splenocyte proliferation were only evident at 2 dpsi (Fig. 7). Following ST re-challenge, splenocyte proliferation in response to both SEAgP and STAgP were significantly higher in those receiving a homologous or heterologous re-challenge compared to age matched primary challenge controls (Fig. 7A and B). Whilst there appeared to be a slight increase in the proliferation of splenocytes from chickens receiving a homologous SE re-challenge at 2 dpsi, this proliferation was not significantly different to those from primary challenge
controls (Fig. 7C and D). At 13 dpsi all of the responses between the various groups were comparable. 4. Discussion The use of a vaccine to protect chickens from a broad range of S. enterica serovars would be a valuable tool for producers of chicken meat and eggs. Challenge in the face of an already existing primary infection (vaccine) presents a problem for data interpretation, where reduced infection may be a result of immune protection or competitive exclusion by the ‘‘vaccinating’’ strain. The immune system of the chickens is still not fully developed and so attenuated strains can persist for long periods in chicks vaccinated at 1-day-old (Barbezange et al., 2000). Current research has focused on potential live attenuated strains such as the Dam and aroA mutants (Cooper et al., 1994; Dueger et al., 2001), which conferred varying levels of protection to challenge with homologous and heterologous virulent
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Fig. 7. Proliferation of splenocytes in response to STAgP and SEAgP. Chickens received a priming challenge with SE or ST, or were left unchallenged at 6 weeks of age, and were (re)-challenged with either ST (A and B) or SE (C and D) at 14 weeks of age. Error bars represent the standard error (n = 5); groups within columns annotated with different letters differ significantly (P < 0.05).
serovars. Vaccination in these studies was undertaken shortly after hatch and therefore reduced infection may result from competitive exclusion rather than immune protection. Indeed studies by Methner et al. (2001) looked specifically at using a competitive exclusion culture alongside a live ST vaccine in 1-day-old chicks; they observed no increase in protection against a virulent strain in those receiving both treatments to those treated with the competitive exclusion culture alone. In the present study we demonstrated the ability of virulent S. enterica serovars to stimulate broadly reactive cellular and humoral immune responses to antigen preparations derived from different S. enterica serovars in immunologically mature chickens. We also observed protection to re-challenge with heterologous or homologous serovars, which corresponded with a high level of cross-reactivity of the induced immune responses to both serovars of S. enterica. It is possible that an ‘‘adjuvant effect’’ (i.e. non-specific stimulation of the immune system by molecules such as Toll-like
receptor agonists) conferred by the priming challenge could contribute to protection against secondary challenge. However, the protection observed in the present study occurred following a challenge 8 weeks after the initial infection and as such is unlikely to be a result of an ‘‘adjuvant effect’’ which would be expected to be much more transient. The high level of cross-reactivity of antibodies to antigen produced from different serovars for both IgA and IgG was not unexpected and is consistent with previous reports (Hassan et al., 1990; Nicholas and Cullen, 1991; Barrow et al., 1992). Birds infected with ST also had higher levels of serum IgG specific for SE antigens than those birds infected with SE. Unlike IgA, IgG is only present in systemic sites; therefore, greater induction of Salmonella-specific IgG by ST could be a response to a higher level of systemic infection. However, levels of Salmonella in the spleen were similar regardless of infecting serovar at 14 dpi, although higher numbers may have been present at
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earlier time points following infection. Cross-reactivity of serum IgA to heterologous serovars was also high, although the IgA response to homologous antigen was consistently highest. Due to the largely enteric nature of the pathogen, IgA is more likely to be involved in any protective immune response after transport into the gut lumen (Lebacq-Verheyden et al., 1972; Leslie and Martin, 1973; Berthelot-Herault et al., 2003). However, whilst the IgA response appears to be more serovar specific than the production of IgG, protection to rechallenge with ST was similar regardless of the priming serovar. The results from the present study indicate a large degree of serovar cross-reactivity in the adaptive immune responses of the chicken to primary infection with either serovar of S. enterica. The data from this study is consistent with previous findings in our laboratory that splenocytes from chickens infected with ST are capable of proliferating in response to SE, SG and SP in addition to ST (unpublished data). Very few studies have examined the capacity of lymphocytes from S. enterica infected chickens to respond to S. enterica antigens ex vivo. Work from our laboratory demonstrated the capacity of lymphocytes to respond to ex vivo antigen stimulation following primary but not secondary infection (Beal et al., 2004a). The data presented here also shows a lack of splenic T cell proliferation following re-infection with either homologous or heterologous serovars, which may be interpreted as the exit of responding T cells from the spleen to sites of infection. The degree of cross-protection acheived by ST to subsequent SE infection was close to that afforded by homologous re-challenge in terms of both systemic and enteric infection. In contrast, SE was less effective in protecting against subsequent ST re-challenge with slightly higher enteric counts than chickens receiving a homologous ST re-challenge. However, the very low numbers of Salmonella in the spleens of both groups indicate that both serovars cross-protect equally well at systemic sites. The capacity of ST and SE to protect against heterologous re-challenge was reflected in the antibody response following secondary infection. High cross-reactivity in serum IgA and IgG against SEAgP and STAgP was evident for all of the infection protocols employed. Whilst high levels of circulating IgA and IgG were observed following challenge and re-challenge, our recent studies suggest that neither B cells or antibody are essential for clearance of a primary or secondary infection with ST (Beal et al., 2006), although no such data exists for primary, heterologous or homologous SE infections.
The data presented here demonstrates that S. enterica from serovar group B can protect against re-challenge with group D and vice versa in immunologically mature chickens. Whilst these data are encouraging for the use of single serovar vaccines to protect mature egg layers against a range of infective serovars, broiler chickens may not develop sufficient immunity in their short production lives for any significant cross-protection to result. Previous work (Beal et al., 2004b) suggests that humoral and cellular immune responses to Salmonella Typhimurium are very low and increase with age (up to 6 weeks). This corresponds with maturation of lymphoid aggregates and immune cell subsets (such as T cells, B cells and heterophils) over the same time period (Befus et al., 1980; Vervelde and Jeurissen, 1993; Gomez Del Moral et al., 1998; Wells et al., 1998; BarShira et al., 2003). Whilst live vaccine strains have shown high levels of protection against infection with heterologous serovars, it has not been shown if this is mediated by an immune response or by competitive exclusion (Dueger et al., 2001, 2003; Methner et al., 2001). The data presented here indicate that there is sufficient immunological cross-protection to merit further investigation of the possibility of developing single serovar live vaccines to protect against both group B and D Salmonella serovars. Acknowledgements We thank the staff of the production and experimental units of the IAH and the Biotechnology and Biological Sciences Research Council and DEFRAHEFCE for funding this research (grant nos. 8/ BFP11365 and VT-0104). References Anonymous, 2003. Postnote 193: Food poisoning. Parliamentary Office of Science and Technology. Baay, M.F., Huis in ’t Veld, J.H., 1993. Alternative antigens reduce cross-reactions in an ELISA for the detection of Salmonella enteritidis in poultry. J. Appl. Bacteriol. 74, 243–247. Bar-Shira, E., Sklan, D., Friedman, A., 2003. Establishment of immune competence in the avian GALT during the immediate post-hatch period. Dev. Comp. Immunol. 27, 147–157. Barbezange, C., Humbert, F., Rose, V., Lalande, F., Salvat, G., 2000. Some safety aspects of salmonella vaccines for poultry: distribution and persistence of three Salmonella typhimurium live vaccines. Avian Dis. 44, 968–976. Barrow, P.A., Berchieri Jr., A., al-Haddad, O., 1992. Serological response of chickens to infection with Salmonella gallinarum– S. pullorum detected by enzyme-linked immunosorbent assay. Avian Dis. 36, 227–236. Barrow, P.A., Bumstead, N., Marston, K., Lovell, M.A., Wigley, P., 2004. Faecal shedding and intestinal colonization of Salmonella
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